Substituted aryl-indole compounds and their kynurenine/kynuramine-like metabolites as therapeutic agents

ABSTRACT

This invention is directed to substituted aryl compounds, which are linked to a substituted indole moiety by various linkers, and the kynurenine/kynuramine-like metabolites of these agents, their preparation and pharmaceutical compositions containing these compounds. This invention further is directed to the pharmaceutical use of the compounds for inhibiting GSK3β kinase and/or modulating N-methyl-D-aspartate (NMDA) channel activities for the treatment of neurodegenerative and other disorders.

FIELD OF THE INVENTION

The present invention relates to a novel family of substituted arylcompounds, pharmaceutical formulations containing them, use of thecompounds in the manufacture of medicaments for treating variousdiseases, and methods of treating these diseases.

BACKGROUND OF THE INVENTION

The present invention relates to novel compounds for inhibiting glycogensynthase kinase-3 (GSK3β) and/or modulators of NMDA channel activitiesand their use in regulating biological conditions mediated by GSK3βactivity and or NMDA channel activity and, more particularly, to the useof such compounds in the treatment of biological conditions such asneurodegenerative diseases, type II diabetes, cancer and affectivedisorders. The present invention further relates to methods of treatingneurodegenerative disorders using GSK3β inhibitors and NMDA modulators.

Synonyms for GSK3β include Tau protein kinase I (TPK I), FA (Factor A)kinase, kinase FA and ATP-citrate lyase kinase (ACLK). GSK3 exists intwo isoforms, i.e. GSK3α and GSK3β, and is a proline-directedserine/threonine kinase originally identified as an enzyme thatphosphorylates glycogen synthase. However, it has been demonstrated thatGSK3β phosphorylates numerous proteins in vitro such as glycogensynthase, phosphatase inhibitor 1-2, the type-II subunit ofcAMP-dependent protein kinase, the G-subunit of phosphatase-1,ATP-citrate lyase, acetyl coenzyme A carboxylase, myelin basic protein,a microtubule-associated protein, a neurofilament protein, an N-CAM celladhesion molecule, nerve growth factor receptor, c-Jun transcriptionfactor, JunD transcription factor, c-Myb transcription factor, c-Myctranscription factor, L-Myc transcription factor, adenomatous polyposiscoli tumor suppressor protein, Tau protein and β-catenin.

GSK3β inhibitors may act to increase the survival of neurons subjectedto aberrantly high levels of excitation induced by the neurotransmitterglutamate (Nonaka, S., et al., Proc. Natl. Acad. Sci. USA, 95(3):2642-7,1998). Glutamate-induced neuronal excitotoxicity is also believed to bea major cause of neurodegeneration associated with acute damage, such asin cerebral ischemia, traumatic brain injury and bacterial infection.Furthermore, it is believed that excessive glutamate signaling is afactor in the chronic neuronal damage seen in diseases such asAlzheimer's, Huntington's, Parkinson's, AIDS associated dementia,amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS) (Thomas,R J., J. Am. Geriatr Soc., 43:1279-89, 1995.

N-methyl-D-aspartate receptors are critical for neuronal plasticity andsurvival, whereas their excessive activation produces excitotoxicity andmay accelerate neurodegeneration. Stimulation of NMDARs in vitro(cultured rat hippocampal or cortical neurons) and in the adult mousebrain in vivo disinhibited GSK3β via protein phosphatase 1(PP1)-mediated dephosphorylation of GSK3β at the serine 9 residue(Szatmari, E., et al, J. Biol. Chem., 280(11):37526-35, 2005).NMDA-triggered GSK3β activation was mediated by NMDAR that contained theNR2B subunit. These data suggest existence of a feedback loop betweenGSK3β and PP1 that results in amplification of PP1 activation by GSK3β.The excessive activation of NR2B-PP1-GSK3β-PP1 circuitry may contributeto the neurodegeneration induced by excessive NMDA. GSK3β inhibitorsmight mimic the action of certain hormones and growth factors, such asinsulin, which use the GSK3β pathway.

GSK3β is considered to be an important player in the pathogenesis ofAlzheimer's disease. GSK-3 was identified as one of the kinases thatphosphorylate Tau, a microtubule-associated protein, which isresponsible for the formation of paired helical filaments (PHF), anearly characteristic of Alzheimer's disease. Apparently, abnormal Tauhyperphosphorylation is the cause for destabilization of microtubulesand PHF formation. Consequently, GSK-3 inhibitors are believed to bepotentially useful for treatment of these and other neurodegenerativedisorders. Indeed, disregulation of GSK-3 activity has been recentlyimplicated in several CNS disorders and neurodegenerative diseases,including schizophrenia (Beasley, C., et al., Neurosci Lett.,302(20):117-20, 2001; Kozlovsky, N., et al., Eur. Neuropsychopharmacol,12:13-25, 2002), stroke, and Alzheimer's disease (AD) (Bhat, R. V. andBudd, S. L., Neurosignals, 11:251-61, 2002; Hernandez, F., et al., J.Neurochem., 83:1529-33, 2002; Lucas, J. J., et al., EMBO J,20:15):27-39, 2001; Mandelkow, E. M., et al., FEBS Lett.,314(21):315-21, 1992).

It thus would be desirable to provide a class of GSK3β inhibitors thatwould be useful in the treatment of diseases mediated through GSK3βactivity such as bipolar disorder (in particular manic depression),diabetes, Alzheimer's disease, leukopenia, FTDP-17 (Fronto-temporaldementia associated with Parkinson's disease), cortico-basaldegeneration, progressive supranuclear palsy, multiple system atrophy,Pick's disease, Niemann Pick's disease type C, Dementia Pugilistica,dementia with tangles only, dementia with tangles and calcification,Down syndrome, myotonic dystrophy, Parkinson's Disease, AmyotrophicLateral Sclerosis (ALS), Parkinsonism-dementia complex of Guam, AIDSrelated dementia, Postencephalic Parkinsonism, prion diseases withtangles, subacute sclerosing panencephalitis, frontal lobe degeneration(FLD), argyrophilic grains disease, subacute sclerotizingpanencephalitis (SSPE) (late complication of viral infections in thecentral nervous system), inflammatory diseases, cancer, dermatologicaldisorders such as baldness, neuronal damage, schizophrenia, pain, inparticular neuropathic pain. GSK3β inhibitors can also be used toinhibit sperm motility and can therefore be used as male contraceptives.

Ions such as glutamate play a key role in processes related to chronicpain and neurotoxicity, primarily by acting through N-methyl-D-aspartatereceptors. Thus, inhibition of such action, by employing ion channelantagonists or negative modulators, can be beneficial in the treatmentand control of CNS diseases. NMDA receptor activity produces synapticplasticity in the central nervous system that affects processes forlearning and memory, including long-term potentiation and long-termdepression (Dingledine R., Crit. Rev. Neurobiol., 4(1):1 96, 1988).However, prolonged activation of NMDA receptor under pathologicalconditions (such as cerebral ischemia and traumatic injury) causesneuronal cell death (Rothman S. M. and Olney J. W., Trends Neurosci.,18(2):57 8, 1995). NMDA receptor-mediated excitotoxicity may contributeto the etiology or progression of several neurodegenerative diseases,such as Parkinson's disease and Alzheimer's disease. Since open channelblockers of NMDA receptors were shown, in the late 1980s, to havepotential for therapy of ischemic stroke, the receptor has beenconsidered an attractive therapeutic target for the development ofneuroprotective agents. Unfortunately, the development of thesecompounds as neuroprotectants is often limited by their psychiatricside-effects associated with their undesired pharmacodynamic propertiessuch as slow dissociation from the receptor (Muir K. W. and Lees K. R.,Stroke, 26(3):503 13, 1995).

Known NMDA antagonists include ketamine, dextromophan, and3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (“CPP”). Althoughthese compounds have been reported (J. D. Kristensen, et al., Pain,51:249 253 (1992); P. K. Eide, et al., Pain, 61:221 228 (1995); D. J.Knox, et al., Anaesth. Intensive Care 23:620 622 (1995); and M. B. Max,et al., Clin. Neuropharmacol. 18:360 368 (1995)) to produce symptomaticrelief in a number of neuropathies including postherpetic neuralgia,central pain from spinal cord injury, and phantom limb pain, widespreaduse of these compounds is precluded by their undesirable side effects.Such side effects at analgesic doses include psychotomimetic effectssuch as dizziness, headache, hallucinations, dysphoria, and disturbancesof cognitive and motor function. Additionally, more severehallucinations, sedation, and ataxia are produced at doses onlymarginally higher than analgesic doses. Thus, it would be desirable toprovide novel NMDA modulators that are absent of undesirable sideeffects or that produce fewer and/or milder side effects.

NMDA receptors are heteromeric assemblies of subunits, of which twomajor subunit families designated NR1 and NR2 have been cloned. Withoutbeing bound by theory, it is generally believed that the variousfunctional NMDA receptors in the mammalian central nervous system(“CNS”) are only formed by combinations of NR1 and NR2 subunits, whichrespectively express glycine and glutamate recognition sites. The NR2subunit family is in turn divided into four individual subunit types:NR2A, NR2B, NR2C and NR2D. Ishii, T., et al., J. Biol. Chem.,268:2836-2843 (1993), and Laurie, D. J., et al., Mol. Brain. Res.,51:23-32 (1997) describe how the various resulting combinations producea variety of NMDA receptors differing in physiological andpharmacological properties such as ion gating properties, magnesiumsensitivity, pharmacological profile, as well as in anatomicaldistribution.

SUMMARY OF THE INVENTION

The invention relates to compounds and their salts having the formula(I):

wherein

each R₁, R₂ and R₃ independently is selected from hydrogen, carboxy,nitro, C₁-C₄ alkylsulfonyl, aminosulfonyl, C₁-C₄ alkyl aminosulfonyl,halogen, cyano, C₁₋₄ alkyl, C₁₋₄ alkoxy, NR′R″, aryl, aryl-C₁₋₄ alkyl,or aryl-C₁₋₄ alkoxy, and each of R′ and R″ is independently H or C₁₋₄alkyl, or R′═R″═ClCH₂CH₂, or NR′R″ constitutes a saturated heterocyclicring containing 3-8 ring members;

X is:—(CH₂)_(n)—Y—wherein Y is: >NH, >C═O, >C═S or none; n is 0-4; any carbon of the—(CH₂)_(n)— may be substituted by 1-2 substituents independentlyselected from among halogen, carboxy, C₁₋₄ alkyl, C₁₋₄ alkoxy, OH, NH₂or acyl,

-   -   Ar is a 3-indole:

or a kynurenine/kynuramine metabolite thereof:

wherein each R₅ independently is hydrogen, halogen, C₁₋₄ alkyl, C₁₋₄alkoxy, OH, NR′R″ as defined above, nitro, aryl, aryl-C₁₋₄ alkyl, oraryl-C₁₋₄ alkoxy;

with the provisos that:

if X is —(CH₂)₂—NH—, Ar is 3-indole, R₁ is 4-methylsulfonyl and R₃ andeach R₅ is hydrogen, then R₂ cannot be 2-nitro;

if X is unsubstituted —(CH₂)₂—NH—, Ar is 3-indole, R₁ is 4-nitro and R₃and each R₅ is hydrogen, then R₂ cannot be 2-bromo;

if X is substituted or unsubstituted —(CH₂)₂—NH— and Ar is 3-indole or2-aminobenzoyl, then R₁ and R₂ cannot be 2,4-dinitro;

and if X is unsubstituted —(CH₂)₂—NH—, Ar is 3-indole, an R₅ is5-methoxy, then R₁ and R₂ cannot be 2,4-dinitro.

In another aspect, the invention provides a pharmaceutical formulationthat comprises at least one pharmaceutically acceptable diluent,preservative, solubilizer, emulsifier, adjuvant, and/or carrier, and atleast one member of the group consisting of the compounds of theinvention as defined above and pharmaceutically acceptable saltsthereof.

In yet another aspect, the invention comprises the administration of aneffective amount of at least one of the compounds of the invention asdefined above and pharmaceutically acceptable salts thereof, for theprevention or treatment of a disease, disorder or biological conditionwhich is mediated by GSK3β activity or NMDA channel activity orassociated with excess GSK3β or NMDA activity.

DETAILED DESCRIPTION OF THE INVENTION

Compounds of the present invention are based on indole and itsmetabolites. The amino acid tryptophan and other indole derivatives suchas melatonin are converted biologically through the “kynurenine pathway”(Beadle, G. W., et al., Proc. Natl. Acad. Sci. USA, 33:155-8, 1947, seeHeidelberger, C., et al., J. Biol. Chem., 179:143, 1949). Over 95% ofall dietary tryptophan is metabolized to kynurenines (Wolf, H., J. Clin.Lab. Invest., 136(Suppl):1-86, 1974). In peripheral tissues,particularly the liver, the indole ring of tryptophan or melatonin ismodified by either tryptophan dioxygenase or indoleamine2,3-dioxygenase, which results in the formation of formylkynurenine orN1-acetyl-N-2-formyl-5-methoxykynuramine (AFMK), respectively. Formylasethen rapidly converts formylkynurenine to L-kynurenine which is the keycompound in the kynurenine pathway (Mehler & Knox 1950) and AFMK toN1-acetyl-5-methoxykynuramine (AMK).

The invention relates to compounds and their salts having the formula(I):

wherein

each of R₁, R₂ and R₃ independently is selected from hydrogen, carboxy,nitro, C₁-C₄ alkylsulfonyl, aminosulfonyl, C₁-C₄ alkyl aminosulfonyl,halogen, cyano, C₁₋₄ alkyl, C₁₋₄ alkoxy, NR′R″, aryl, aryl-C₁₋₄ alkyl,or aryl-C₁₋₄ alkoxy, and each of R′ and R″ is independently H or C₁₋₄alkyl, or R′═R″═ClCH₂CH₂, or NR′R″ constitutes a saturated heterocyclicring containing 3-8 ring members;

X is:—(CH₂)_(n)—Y—wherein Y is: >NH, >C═O, >C═S or none; n is 0-4; any carbon of the—(CH₂)_(n)— may be substituted by 1-2 substituents independentlyselected from among halogen, carboxy, C₁₋₄ alkyl, C₁₋₄ alkoxy, OH, NH₂or acyl,Ar is a 3-indole:

or a kynurenine/kynuramine metabolite thereof:

wherein each R₅ independently is hydrogen, halogen, C₁₋₄ alkyl, C₁₋₄alkoxy, OH, NR′R″ as defined above, nitro, aryl, aryl-C₁₋₄ alkyl, oraryl-C₁₋₄ alkoxy;with the provisos that:

if X is —(CH₂)₂—NH—, Ar is 3-indole, R₁ is 4-methylsulfonyl and R₃ andeach R₅ is hydrogen, then R₂ cannot be 2-nitro;

if X is unsubstituted —(CH₂)₂—NH—, Ar is 3-indole, R₁ is 4-nitro and R₃and each R₅ is hydrogen, then R₂ cannot be 2-bromo; and

if X is substituted or unsubstituted —(CH₂)₂—NH— and Ar is 3-indole or2-aminobenzoyl, then R₁ and R₂ cannot be 2,4-dinitro;

and if X is unsubstituted —(CH₂)₂—NH—, Ar is 3-indole, an R₅ is5-methoxy, then R₁ and R₂ cannot be 2,4-dinitro.

In preferred embodiments, Y is >NH, each of R₁, R₂ and R₃ isindependently selected from hydrogen, carboxy, nitro, C₁-C₄alkylsulfonyl, halogen and cyano, and each R₅ independently is selectedfrom hydrogen and C₁₋₄ alkoxy.

Preferred compounds within the generic class of compounds set forthabove include2-(2-aminobenzoyl)-N-2-nitro-4-methylsulfonyl-phenylethylamine;N-(4-methylsulfonyl-2-nitrophenyl)-5-methoxytryptamine;N-(2-bromo-4-nitrophenyl)-5-methoxytryptamine;N-(4-bromo-2-nitrophenyl)-5-methoxytryptamine;N-(2-cyano-4-nitrophenyl)-5-methoxytryptamine;2-(2-aminobenzoyl)-N-2-bromo-4-nitro-phenylethylamine;2-(2-aminobenzoyl)-N-2-nitro-4-bromo-phenylethylamine;2-(2-aminobenzoyl)-N-2-nitro-4-cyano-phenylethylamine;N-(2-nitrophenyl)-tryptamine; N-(4-carboxy-2-nitrophenyl)-tryptamine;N-(2-carboxy-4-nitrophenyl)-tryptamine;N-(2-nitrophenyl)-5-methoxytryptamine;N-(4-carboxy-2-nitrophenyl)-5-methoxytryptamine;N-(2-carboxy-4-nitrophenyl)-5-methoxytryptamine;N-(2-cyano-4-nitrophenyl)-tryptamine;N-(2-nitro-4-bromophenyl)-tryptamine, N-(3,4-dicyanophenyl)-tryptamine,N-(3,4-dicyanophenyl)-5-methoxytruptamine and2-(2-aminobenzoyl)-N-2-nitrophenylethylamine.

Particularly preferred compounds includeN-(4-methylsulfonyl-2-nitrophenyl)-5-methoxytryptamine;N-(2-nitrophenyl)-5-methoxytryptamine;N-(2-cyano-4-nitrophenyl)-tryptamine;2-(2-aminobenzoyl)-N-2-nitro-4-methylsulfonyl-phenylethylamine; andN-(2-nitrophenyl)-tryptamine.

In another aspect, the invention provides a pharmaceutical formulationthat comprises at least one pharmaceutically acceptable diluent,preservative, solubilizer, emulsifier, adjuvant, and/or carrier, and atleast one member of the group consisting of the compounds of theinvention as defined above and pharmaceutically acceptable saltsthereof.

A pharmaceutical formulation according to the invention is preferablycharacterized by at least one of the following features:

(i) it is adapted for oral, rectal, parenteral, transbuccal, topical,intrapulmonary (e.g. by inhalation), intranasal or transdermaladministration;

(ii) it is in unit dosage form, each unit dosage comprising an amount ofat least one compound of formula (I) which is within the range of about0.001-about 100 mg/kg;

(iii) it is a controlled release formulation, wherein at least onecompound of formula (I) is released at a predetermined controlled rate.

The amount of a compound of formula (I) useful in treating a disease ordisorder can vary with the nature and severity of the condition to betreated, the particular method of administration selected, the frequencyof administration, the age, sex, weight and general condition of thepatient and other factors evident to those of skill in the art.Generally, if the unit dosage is to be administered orally, a dosewithin the range of about 0.01 mg/kg-about 50 mg/kg daily, preferablywithin the range of about 0.05 mg-about 10 mg/kg, is effective. A morepreferred dosage for oral administration is within the range of about0.5-about 10 mg/kg daily. If the compound is to be administeredparenterally or transdermally, a unit dosage within the range of about0.005-about 15 mg/kg generally is desirable.

For oral administration, the pharmaceutical formulations may be utilizedas, e.g., tablets, orally disintegrating tablets, capsules, emulsions,solutions, syrups or suspensions. For parenteral administration, theformulations can be utilized as ampoules, or otherwise as suspensions,solutions or emulsions in aqueous or oily vehicles. The need forsuspending, stabilizing and/or dispersing agents will, of course, takeaccount of the fact of the solubility or otherwise of the activecompounds, in the vehicles that are used in the particular embodiments.The formulations additionally can contain physiologically compatiblepreservatives and antioxidants. In the formulations for topicalapplication, e.g. creams, lotions or pastes, the active ingredient canbe mixed with conventional oleaginous or emulsifying excipients.

The pharmaceutical formulations also can be utilized as suppositorieswith conventional suppository bases such as cocoa butter or otherglycerides. Alternatively, the formulations can be made available in adepot form, which will release the active composition slowly in thebody, over a pre-selected time period.

The compounds of the invention also can be administered by usingconventional transbuccal, intranasal, intrapulmonary or transdermaldelivery systems.

The compounds of formula (I) or their salts can be administered incombination with other therapeutic agents, especially compounds that actas anxiolytics, tranquilizers, analgesics, mood stabilizers,anti-Parkinson's agents (dopaminergic and non-dopaminergic drugs),anti-Alzheimer's drugs or anti-diabetic agents. “In combination” as usedherein is intended to mean either that the compounds of the inventionare physically combined with one or more additional therapeutic agentsor that they are administered in separate physical forms butsufficiently close in time that both act within the body within a giventime period. Examples of suitable anxiolytics which can be administeredin combination with the compounds of formula I include flunitrazepam,diazepam and alprazolam; suitable tranquilizers include clonazepam,zolpidem, trazodone and melatonin; suitable analgesics include aspirin,ibuprofen and diclofenac; suitable mood stabilizers include lithium,sodium valproate and carbamazepine; suitable anti-Parkinson's agentsinclude levodopa/carbidopa, cabergolline, pergolide, pramipexole,ropinirol, entacapone (COMT inhibitor), selegiline and rasagiline (MAO-Binhibitors); and suitable anti-diabetic agents include metformin,acarbose and glipizide. These known therapeutic agents can be physicallycombined with the compounds of the present invention or administered incombination with the compounds of the present invention but in separatephysical form.

The compounds of formula I and their salts are administered to inhibitGSK3β activity or NMDA channel activity in animals or humans. Moreparticularly, the compounds can be administered to prevent or to treatdiseases, disorders or conditions which are mediated through GSK3βactivity or NMDA channel activity or associated with excess GSK3βactivity or NMDA channel activity. Such diseases, disorders andconditions, include central nervous system (CNS) disorders and traumasand neurodegenerative diseases, such as bipolar disorder (particularlymanic-depressive disorder), Alzheimer's disease, Parkinson's disease,FTDP-17 (frontal-temporal dementia associated with Parkinson's disease),cortico-basal degeneration, progressive supranuclear palsy, multiplesystem atrophy, Pick's disease, Niemann Pick's disease type C, DementiaPugilistica, dementia with tangles only, dementia with tangles andcalcification, Parkinsonism-dementia complex of Guam, AIDS-relateddementia, postencepalic Parkinsonism, prion diseases with tangles,Amyotrophic Lateral Sclerosis (ALS) subacute sclerosing panencephalitis,frontal lobe degeneration (FLD), argyrophilic grains disease, subacutesclerotizing panencephalitis (SSPE) (late complication of viralinfections in the central nervous system), neuronal damage andschizophrenia; diabetes; leukopenia; Down Syndrome; myotonic dystrophy;inflammatory diseases; cancer and other proliferative disorders;dermatological disorders, such as baldness; cancer; pain, includingneuropathic pain and chronic pain; migraines, psychiatric diseases, suchas depression; anxiety; and stroke.

The invention is further illustrated by the following examples which areprovided for illustrative purposes only and are not intended to belimiting.

EXAMPLE 1 2-(2-aminobenzoyl)-N-2-nitro-4-methylsulfonyl-phenylethylamine

General procedure for the synthesis of2-(2-aminobenzoyl)-N-2-nitro-4-methylsulfonyl-phenylethylamine

In a 100 ml three-necked round-bottom flask kept under an argonatmosphere, 250 mg (1.14 mmoles, 1 eq) ofmethyl-4-fluoro-3-nitrobenzensulfone were dissolved in 20 ml of ethanol.Kynuramine dihydrobromide 371 mg (1 eq) was then added under magneticstirring in one portion. After 15 minutes Na₂CO₃ 326 mg (3 eq) was addedto the reaction.

The reaction course was followed by HPLC-MS that, after 6 hours, showedcomplete conversion. The yellow precipitate was then collected byfiltration, washed with water and cold EtOH and then dried under vacuumat 40° C.

The desired product was recovered as a yellow solid (300 mg). 1H NMR(DMSO-d₆, 400 MHz) δ 3.20 (s, 3H, SO₂CH₃), 3.38 (br t, J=6.8 Hz, 2H,NHCH₂CH₂), 3.77-3.81 (m, 2H, NHCH₂CH₂), 6.51-6.55 (m, 1H, aromatic H),6.76 (dd, J=1.2 Hz, J₂=8.4 Hz, 1H, aromatic H), 7.22-7.27 (m, 3H, 1aromatic H+NH₂), 7.35 (d, J=9.6 Hz, 1H, aromatic H), 7.76 (dd, J₁=1.4Hz, J₂=8.4 Hz, 1H, aromatic H), 7.92 (dd, J₁=2.1 Hz, J₂=9.0 Hz, 1H,aromatic H), 8.49 (d, J=2.1 Hz, 1H, aromatic H), 8.72 (br t, J=5.9 Hz,1H, NHCH₂CH₂).

EXAMPLE 2 N-(4-methylsulfonyl-2-nitrophenyl)-5-methoxytryptamine

General procedure for the synthesis ofN-(4-methylsulfonyl-2-nitrophenyl)-5-methoxytryptamine

In a 100 ml three-necked round-bottom flask kept under an argonatmosphere, 483 mg (2.20 mmoles, 1 eq) ofmethyl-4-fluoro-3-nitrobenzensulfone were dissolved in 40 ml of ethanol.5-methoxytryptamine hydrochloride 500 mg (1 eq) was then added undermagnetic stirring in one portion. After 15 minutes Na₂CO₃ 466 mg (2 eq)was added to the reaction. The reaction was heated to 50° C. using anoil bath. The reaction course was followed by HPLC-MS that, after 3hours, showed complete conversion. The orange precipitate was collectedby filtration, washed with water and cold EtOH and then dried undervacuum at 40° C. The desired product was recovered as an orange solid(350 mg).

¹H NMR (DMSO-d₆, 400 MHz) δ 3.05 (t, J=7.1 Hz, 2H, NHCH₂CH₂), 3.20 (s,3H, SO₂CH₃), 3.71-3.75 (m, 5H, OCH₃+NHCH₂CH₂), 6.71 (dd, J₁=2.6 Hz,J₂=8.8 Hz, 1H, aromatic H), 7.07 (d, J=2.4 Hz, 1H, aromatic H),7.22-7.24 (m, 2H, aromatic H), 7.28 (d, J=9.0 Hz, 1H, aromatic H), 7.89(dd, J₁=2.0 Hz, J₂=8.9 Hz, 1H, aromatic H), 8.47 (d, J=2.6 Hz, 1H,aromatic H), 8.64 (br t, J=5.7 Hz, 1H, NHCH₂CH₂), 10.73 (br s, 1H, NH).

EXAMPLE 3 N-(2-bromo-4-nitrophenyl)-5-methoxytryptamine

EXAMPLE 4 N-(4-bromo-2-nitrophenyl)-5-methoxytryptamine

EXAMPLE 5 N-(2-cyano-4-nitrophenyl)-5-methoxytryptamine

General procedure for the synthesis ofN-(2-bromo-4-nitrophenyl)-5-methoxytryptamine,N-(4-bromo-2-nitrophenyl)-5-methoxytryptamine andN-(2-cyano-4-nitrophenyl)-5-methoxytryptamine

1 equivalent of 1-fluoro-2R₁-4R₂-benzene was reacted in ethanol, at roomtemperature, with 1 equivalent of 5-methoxytryptamine, to yield thedesired product, as follows:

(N-(2-bromo-4-nitrophenyl)-5-methoxytryptamine): R₁=Br, R₂=NO₂; reactiontime 3 h; yield referred to chromatographed product: 70%

(N-(4-bromo-2-nitrophenyl)-5-methoxytryptamine): R₁=NO₂, R₂=Br; reactiontime 3 h; yield referred to isolated product (collected by filtration):50%

N-(2-cyano-4-nitrophenyl)-5-methoxytryptamine): R₁=CN, R₂=NO₂; reactiontime 3 h; yield referred to isolated product (collected by filtration):50%

NMR spectra of compounds N-(2-bromo-4-nitrophenyl)-5-methoxytryptamine,N-(4-bromo-2-nitrophenyl)-5-methoxytryptamine andN-(2-cyano-4-nitrophenyl)-5-methoxytryptamineN-(2-bromo-4-nitrophenyl)-5-methoxytryptamine

¹H NMR (DMSO-d₆, 400 MHz) δ 2.96 (t, J=7.7 Hz, 2H, CH₂), 3.54-3.59 (m,2H, CH₂—NH), 3.74 (s, 3H, OCH₃), 6.62 (br t, J=5.8 Hz, 1H, CH₂—NH), 6.70(dd, J₁=2.5 Hz, J₂=8.7 Hz, 1H, aromatic H), 6.83 (d, J=9.2 Hz, 1H,aromatic H), 7.03 (d, J=2.2 Hz, 1H, aromatic H), 7.18-7.22 (m, 2H,aromatic H), 8.04 (dd, J₁=2.2 Hz, J₂=9.2 Hz, 1H, aromatic H), 8.25 (d,J=2.5 Hz, 1H, aromatic H), 10.69 (br s, 1H, NH).

N-(4-bromo-2-nitrophenyl)-5-methoxytryptamine

¹H NMR (DMSO-d₆, 400 MHz) δ 3.01 (t, 2H, J=6.9 Hz, CH₂), 3.59-3.64 (m,2H, CH₂—NH), 3.73 (s, 3H, OCH₃), 6.70 (dd, J₁=2.8 Hz, J₂=8.7 Hz, 1H,aromatic H), 7.03-7.07 (m, 2H, aromatic H), 7.19-7.22 (m, 2H, aromaticH), 7.60 (dd, J=2.1 Hz, J₂=9.6 Hz, 1H, aromatic H), 8.11 (d, J=2.8 Hz,1H, aromatic H), 8.20 (br t, J=5.6 Hz, 1H, CH₂—NH), 10.71 (br s, 1H,NH).

N-(2-cyano-4-nitrophenyl)-5-methoxytryptamine

¹H NMR (DMSO-d₆, 400 MHz) δ 2.97 (t, 2H, J=7.4 Hz, CH₂), 3.58-3.63 (m,2H, CH₂—NH), 3.76 (s, 3H, OCH₃), 6.71 (dd, J₁=2.5 Hz, J₂=8.8 Hz, 1H,aromatic H), 6.93 (d, J=9.6 Hz, 1H, aromatic H), 7.04 (d, J=2.2 Hz, 1H,aromatic H), 7.17-7.23 (m, 2H, aromatic H), 7.59 (br t, J=6.0 Hz, 1H,CH₂—NH), 8.15 (dd, J₁=3.0 Hz, J₂=9.4 Hz, 1H, aromatic H), 8.41 (d, J=2.9Hz, 1H, aromatic H), 10.70 (br s, 1H, NH).

EXAMPLE 6 2-(2-aminobenzoyl)-N-2-bromo-4-nitro-phenylethylamine

EXAMPLE 7 2-(2-aminobenzoyl)-N-2-nitro-4-bromo-phenylethylamine

EXAMPLE 8 2-(2-aminobenzoyl)-N-2-nitro-4-cyano-phenylethylamine

General procedure for the synthesis of2-(2-aminobenzoyl)-N-2-bromo-4-nitro-phenylethylamine,2-(2-aminobenzoyl)-N-2-nitro-4-bromo-phenylethylamine and2-(2-aminobenzoyl)-N-2-nitro-4-cyano-phenylethylamine

3×125 mg (3×1 equiv) of kynuramine dihydrobromide were dissolved underan argon atmosphere in 3×1 ml of absolute ethanol in three differentflasks of a Carousel parallel synthesizer. Triethylamine (3×0.1 ml, 3×2equiv) was also added in each flask. 2-Bromo-1-fluoro-4-nitrobenzene (85mg, 1 equiv), 4-bromo-1-fluoro-2-nitrobenzene (85 mg, 1 equiv) and2-fluoro-5-nitrobenzonitrile (65 mg, 1 equiv) were then addedrespectively in one of the three parallel flasks (A, B and C) and theobtained mixtures were allowed to react at room temperature undermagnetic stirring. The course of the reactions was followed by TLC(dichloromethane as the eluent). Reactions A, B and C were all completedafter 16 hours. The three reaction mixtures were then concentrated underreduced pressure and the resulting residues were purified by columnchromatography on silica gel (approx. 10 grams) by using dichloromethaneas the eluent.

2(2-aminobenzoyl)-N-2-bromo-4-nitro-phenylethylamine was obtained as ayellow solid in 30% yield,2(2-aminobenzoyl)-N-2-nitro-4-bromo-phenylethylamine was collected as anorange solid in 40% yield and2(2-aminobenzoyl)-N-2-nitro-4-cyano-phenylethylamine was isolated as ayellow solid in 40% yield.

EXAMPLE 9 N-(2-Nitrophenyl)-tryptamine

Procedure:

In a 250 ml round bottom flask DMF (1 eq.), tryptamine (1 eq.),2-nitrofluorobenzene (1 eq.) were taken and stirred for 10 min. Thenpotassium carbonate (1.1 eq.) was added at room temperature. Thestirring was continued for 2 hours. TLC was monitored. The reactionmixture was poured into ice water and stirred for 15 min. The resultantsolid was filtered and washed with water. The crude material wascrystallized from methanol. The yield was 60%.

NMR: (CDCl₃) δ 3.2 (t, 2H, CH2), 3.6 (t, 2H, CH₂NH), 6.6 (t, 1H, 4′-H),6.8 (d, 1H, 7-H), 7.1-7.3 (m, 3H, 2-H, 5-H, 6-H), 7.4 (m, 2H, 4-H,6′-H), 7.6 (d, 1H, 5′-H) 8.1 (m, 3H, 3′-H, 2×NH).

EXAMPLE 10 N-(4-Carboxy-2-nitrophenyl)-tryptamine

Procedure:

In a 250 ml round bottom flask DMF (10 eq.), tryptamine (1 eq.), and4-carboxy-2-nitrofluorobenzene (1 eq.) were added and stirred for 10min. Then potassium carbonate (2.5 eq.) was added at room temperature.The stirring was continued for 2 hrs. TLC was monitored. The reactionmixture was poured into ice water, neutralized with acetic acid to pH=5and stirred for 15 min. The resultant solid was filtered and washed withwater. The crude material was crystallized from toluene. The yield was50%.

NMR: (CDCl3) δ 3.3 (t, 2H, CH2), 3.5 (t, 2H, CH2NH), 6.8 (d, 1H, 6′-H),7.2 (m, 2H, 5-H, 6-H), 7.4 (d, 1H, 7-H), 7.6 (d, 1H, 4-H), 8.0 (d, 1H,5′-H), 8.4 (bs, 1H, NH), 8.6 (s, 1H, NH), 8.8 (s, 1H, 3′-H).

EXAMPLE 11 N-(2-Carboxy-4-nitrophenyl)-tryptamine

Procedure:

In a 250 ml round bottom flask DMF (10 eq.), tryptamine (1 eq.),2-carboxy-4-nitrofluorobenzene (1 eq.) were added and stirred for 10min. Then potassium carbonate (2.5 eq.) was added at room temperature.The stirring was continued for 2 hrs. TLC was monitored. The reactionmixture was poured into ice water, neutralized with acetic acid to pH=5and stirred for 15 min. The resultant solid was filtered and washed withwater. The crude material was crystallized from toluene. The yield was50%.

NMR: (CDCl3) δ 3.1 (t, 2H, CH₂), 3.6 (t, 2H, CH₂NH), 6.7 (d, 1H, 5′-H),7.0 (m, 3H, 2-H, 5-H, 6-H), 7.4 (d, 1H, 7-H), 7.5 (d, 1H, 4-H), 8.1 (d,1H, 4′-H), 8.8 (d, 1H, 3′-H), 8.9 (bs, 1H, NH), 10.4 (s, 1H, NH).

EXAMPLE 12 N-(2-Nitrophenyl)-5-methoxytryptamine

Procedure:

In a 250 ml round bottom flask DMF (10 eq.), 5-methoxy-tryptamine (1eq.) and 2-nitrofluorobenzene (1 eq.) were added and stirred for 10 min.Then potassium carbonate (1.1 eq.) was added at room temperature. Thestirring was continued for 2 hrs. TLC was monitored. The reactionmixture was poured into ice water and stirred for 15 min. The resultantsolid was filtered and washed with water. The crude material wascrystallized from methanol. The yield was 60%.

NMR: (CDCl3) δ 3.2 (t, 2H, CH₂), 3.6 (t, 2H, CH₂NH), 3.8 (s, 3H, OCH₃),6.6 (t, 1H, 4′-H), 6.8 (d, 2H, 6-H, 7-H), 7.0 (d, 1H, 4-H), 7.1 (s, 1H,2-H), 7.3 (d, 1H, 6′-H), 7.9 (bs, 1H, NH), 8.2 (m, 2H, 3′-H, NH).

EXAMPLE 13 N-(4-Carboxy-2-nitrophenyl)-5-methoxytryptamine

Procedure:

In a 250 ml round bottom flask DMF (10 eq.), 5-methoxytryptamine (1 eq.)and 4-carboxy-2-nitrofluorobenzene (1 eq.) were added and stirred for 10min. Then potassium carbonate (2.5 eq.) was added at room temperature.The stirring was continued for 2 hrs. TLC was monitored. The reactionmixture was poured into ice water, neutralized with acetic acid to pH=5and stirred for 15 min. The resultant solid was filtered and washed withwater. The crude material was crystallized from toluene. The yield was40%.

NMR: (CDCl₃) δ 3.5 (m, 4H, 2×CH₂), 3.8 (s, 3H, OCH₃), 6.7 (d, 1H, 6′-H),6.9 (bs, 1H, 7-H), 6.95 (s, 1H, 2H), 7.1 (s, 1H, 4-H), 7.3 (d, 1H,5′-H), 8.0 (bs, 1H, 6-H), 8.4 (bs, 1H, 3′-H), 8.8 (s, 1H, NH), 10.4 (s,1H, NH).

EXAMPLE 14 N-(2-Carboxy-4-nitrophenyl)-5-methoxytryptamine

Procedure:

In a 250 ml round bottom flask DMF (10 eq.), 5-methoxytryptamine (1eq.), 2-carboxy-4-nitrofluorobenzene (1 eq.) were added and stirred for10 min. Then potassium carbonate (2.5 eq.) was added at roomtemperature. The stirring continued for 2 hrs. TLC was monitored. Thereaction mixture was poured into ice water, neutralized with acetic acidto pH=5 and stirred for 15 min. The resultant solid was filtered andwashed with water. The crude material was crystallized from toluene. Theyield was 40%.

NMR: (CDCl3) δ 3.1 (t, 2H, CH₂), 3.6 (t, 2H, NH), 3.8 (s, 3H, OCH₃), 6.6(m, 2H, 4-H, 5′-H), 6.7 (m, 5H, Ar—H), 8.1 (d, 1H, 5-H), 8.7 (d, 1H,3′-H), 8.9 (bs, 1H, NH), 10.5 (s, 1H, NH).

EXAMPLE 15 N-(2-cyano-4-nitrophenyl)-tryptamine

EXAMPLE 16 N-(2-nitro-4-bromophenyl)-tryptamine

General procedure for the synthesis ofN-(2-nitro-4-bromophenyl)-tryptamine andN-(2-cyano-4-nitrophenyl)-tryptamine

2×500 mg (2×1 equiv) of tryptamine were dissolved under an argonatmosphere in 2×2 ml of absolute ethanol in three different flasks of aCarousel parallel synthesizer. 4-bromo-1-fluoro-2-nitrobenzene (690 mg,1 equiv) and 2-fluoro-5-nitrobenzonitrile (520 mg, 1 equiv) were added,respectively, in one of the two parallel flasks (A and B) and theobtained mixtures were allowed to react at room temperature undermagnetic stirring. The course of the reactions was followed by TLC(dichloromethane as the eluent). Reactions A and B were completed after8 and 2 hours, respectively. The two mixtures were then diluted with ca.15 ml of diethyl ether and the resulting precipitates were collected byfiltration and washed with additional Et₂O. TLC analyses showed in allprecipitates residual traces of starting materials, thus each reactionmixture was purified by column chromatography on silica gel (approx. 20grams). A mixture of petroleum ether/dichloromethane (8:2) was useduntil the starting nitroaromatic derivatives were eluted; subsequently,the target products were eluted by using dichloromethane.N-(2-nitro-4-bromophenyl)-tryptamine was collected as a red solid in 55%yield and finally N-(2-cyano-4-nitrophenyl)-tryptamine was obtained as ayellow solid in 40% yield.

NMR spectra of compounds, N-(2-nitro-4-bromophenyl)-tryptamine andN-(2-cyano-4-nitrophenyl)-tryptamineN-(2-nitro-4-bromophenyl)-tryptamine

¹H NMR (DMSO-d₆, 400 MHz) δ 3.07 (t, 2H, J=6.9 Hz, CH₂), 3.62-3.68 (m,2H, CH₂—NH), 6.98 (t, J=6.9 Hz, 1H, aromatic H), 7.06-7.10 (m, 2H,aromatic H), 7.26 (br s, 1H, aromatic H), 7.35 (d, J=8.0 Hz, 1H,aromatic H), 7.58 (d, J=8.3 Hz, 1H, aromatic H), 7.62 (dd, J₁=2.2 Hz,J₂=8.8 Hz, 1H, aromatic H), 8.13 (d, J=2.2 Hz, 1H, aromatic H), 8.20 (brt, J=5.4 Hz, 1H, CH₂—NH), 10.87 (br s, 1H, NH).

N-(2-cyano-4-nitrophenyl)-tryptamine

¹H NMR (DMSO-d₆, 400 MHz) δ 3.01 (t, 2H, J=7.2 Hz, CH₂), 3.59-3.64 (m,2H, CH₂—NH), 6.93 (d, J=9.6 Hz, 1H, aromatic H), 6.99 (t, J=7.4 Hz, 1H,aromatic H), 7.08 (t, J=6.9 Hz, 1H, aromatic H), 7.21 (br s, 1H,aromatic H), 7.34 (d, J=8.0 Hz, 1H, aromatic H), 7.55-7.60 (m, 2H,aromatic H+CH₂—NH), 8.15 (dd, J₁=2.1 Hz, J₂=9.5 Hz, 1H, aromatic H),8.39 (d, J=2.1 Hz, 1H, aromatic H), 10.85 (br s, 1H, NH).

EXAMPLE 17 N-(3,4-dicyanophenyl)-tryptamine

General procedure for the synthesis of N-(3,4-dicyanophenyl)-tryptamine

Under an argon atmosphere, a 100 ml three-necked round-bottom flask wascharged with tryptamine (1.10 g, 1 equiv.) dissolved in EtOH (12 ml). Tothe solution 4-fluoro-phtahalonitrile (1.00 g, 1 equiv.) was then addedin one portion. The resulting mixture was allowed to react undermagnetic stirring for 25 h at room temperature. The reaction course wasfollowed by TLC and HPLC-MS. The solvent was then removed by rotaryevaporation and the crude product was chromatographed over a silica gelcolumn by eluting with dichloromethane. The product was recovered as anoff-white solid (880 mg, yield 35%).

¹H NMR (CDCl₃, 400 MHz) δ 3.13 (t, J=6.3 Hz, 2H, CH₂CH₂NH), 3.51-3.56(m, 2H, CH₂CH₂NH), 4.54 (br t, J=5.3 Hz, 1H, CH₂CH₂NH), 6.69 (dd, J₁=2.3Hz, J₂=8.6 Hz, 1H, aromatic H), 6.79 (d, J=2.5 Hz, 1H, aromatic H), 7.06(d, J=2.3 Hz, 1H, aromatic H), 7.14-7.18 (m, 1H, aromatic H), 7.23-7.27(m, 1H, aromatic H), 7.41 (br d, J=8.1 Hz, 1H, aromatic H), 7.46 (d,J=8.8 Hz, 1H, aromatic H), 7.57 (br d, J=8.1 Hz, 1H, aromatic H), 8.08(br s, 1H, NH).

EXAMPLE 18 N-(3,4-dicyanophenyl)-5-methoxytryptamine

General procedure for the synthesis ofN-(3,4-dicyanophenyl)-5-methoxytryptamine

Under an argon atmosphere, a 100 ml three-necked round bottom flask wascharged with 5-methoxytryptamine (1.33 g, 1 equiv.) dissolved in hotEtOH (20 ml). The solution was then cooled to room temperature and4-fluoro-phtahalonitrile (1.00 g, 1 equiv.), was added in one portion.The resulting mixture was allowed to react under magnetic stirring for20 h at room temperature. The reaction course was followed by TLC andHPLC-MS. The solvent was then removed by rotary evaporation and thecrude product was chromatographed over a silica gel column by elutingwith dichloromethane. The product was recovered as a white solid (490mg, yield 22%).

¹H NMR (CDCl₃, 400 MHz) δ 3.09 (t, J=6.6 Hz, 2H, CH₂CH₂NH), 3.50-3.54(m, 2H, CH₂CH₂NH), 3.85 (s, 3H, OCH₃), 4.55 (br t, J=5.1 Hz, 1H,CH₂CH₂NH), 6.69 (dd, J₁=2.3 Hz, J₂=8.8 Hz, 1H, aromatic H), 6.80 (d,J=2.8 Hz, 1H, aromatic H), 6.90 (dd, J₁=2.0 Hz, J₂=8.8 Hz, 1H, aromaticH), 6.98 (d, J=2.9 Hz, 1H, aromatic H), 7.03 (d, J=2.3 Hz, 1H, aromaticH), 7.30 (d, J=8.8 Hz, 1H, aromatic H), 7.47 (d, J=8.8 Hz, 1H, aromaticH), 7.97 (br s, 1H, NH).

EXAMPLE 19 2-(2-aminobenzoyl)-N-2-nitrophenylethylamine

2 ml of 2-nitro-fluorobenzene were reacted in 20 ml DMF, at roomtemperature, with 5 g of kynuramine and 3 g of potassium carbonate, toyield the desired product; reaction time was 2 h. The reaction mixturewas placed in 250 ml of water and stirred. It was extracted intoethylacetate (2×100 ml), ethylacetate layer was washed twice with water(50 ml), dried with sodium sulphate and the solvent was distilled off.The crude material was purified by column chromatography run withethylacetate and hexane mixture (1:9).

Yield referred was 500 mg.

¹H NMR (DMSO-d₆, 500 MHz) δ 3.35 (t, 2H, NHCH₂CH₂, J=6.6 Hz), 3.69 (q,2H, NHCH₂CH₂, J=6.4 Hz), 6.52 (t, 1H, aromatic, J=7.6 Hz), 6.68 (t, 1H,aromatic, J=7.8 Hz), 6.75 (d, 1H, aromatic, J=8.3 Hz), 7.13 (d, 1H,aromatic, J=8.7 Hz), 7.23 (m, 3H, 1 aromatic H+NH₂), 7.55 (t, 1H,aromatic, J=7.8 Hz), 7.77 (d, 1H, aromatic, J=8.1 Hz), 8.06 (d, 1H,aromatic, J=8.7 Hz), 8.23 (t, 1H, NHCH₂CH₂, J=5.6 Hz)

Biological Testing of Compounds of the Invention

Experiment 1:

Evaluation of GSK3β Activity:

Compounds were evaluated for inhibition against purified GSK3β. GSK3βwas expressed in and purified from insect Sf9 cells. Compounds (10 μM)were assayed; following a 1/100 dilution of the enzyme in 1 mg/ml BSA,10 mM DTT, with 5 μl of 40 μM GS-2 peptide as a substrate, in a buffer,in the presence of 15 μM [γ-³²P]ATP (3000 Ci/mmol; 1 mCi/ml) in a finalvolume of 30 μl. After 30-min incubation at 30° C., 25-μl aliquots ofsupernatant were spotted onto 2.5×3 cm pieces of Whatman P81phosphocellulose paper, and, 20 s later, the filters were washed fivetimes (for at least 5 min each time) in a solution of 10 ml ofphosphoric acid/liter of water. The wet filters were counted in thepresence of 1 ml of scintillation fluid. Table 1 presents the GSK3βactivity inhibition by compounds of the present patent application.

TABLE 1 GSK3β Activity Tested Substance Inhibition IC50N-(2-Nitrophenyl)-tryptamine  9.9 μM N-(2-cyano-4-nitrophenyl)- 12.7 μMtryptamine N-(2-Nitrophenyl)-5- 14.1 μM methoxytryptamineN-(2-Carboxy-4-nitrophenyl)- 14.2 μM tryptamineN-(3,4-dicyanophenyl)-tryptamine 14.8 μM N-(3,4-dicyanophenyl)-5- 16.8μM methoxytryptamine 2-(2-aminobenzoyl)-N-2- 18.5 μMnitrophenylethylamine N-(4-Carboxy-2-nitrophenyl)-5- 21.7 μMmethoxytryptamine 2-(2-aminobenzoyl)-N-2-nitro-4- 29.3 μMcyano-phenylethylamineThis experiment revealed that N-(2-Nitrophenyl)-tryptamine,N-(2-cyano-4-nitrophenyl)-tryptamine,N-(2-nitrophenyl)-5-methoxytryptamine,N-(2-carboxy-4-nitrophenyl)-tryptamine,N-(3,4-dicyanophenyl)-tryptamine,N-(3,4-dicyanophenyl)-5-methoxytryptamine,N-(4-carboxy-2-nitrophenyl)-5-methoxytryptamine and2-(2-aminobenzoyl)-N-2-nitro-4-cyano-phenylethylamine have a significantinhibiting activity on GSK3β activity.Experiment 2:Evaluation of Anti-Parkinsonian Activity Using MPTP-Treated Micewith/without a Sub Threshold Dose of L-Dopa

Animals: six month old male C57 BL/6 mice, weighing 22-25 g were used.Following arrival at the laboratory, the mice were allowed toacclimatize for two weeks in a room with controlled temperature (21±1°C.), and a constant light-dark schedule (12 hr on/12 hr off, lights onbetween 06.00 and 18.00 hrs). Free access to food and water wasmaintained throughout. They were housed in groups of 12 animals andtested only during the hours of light (08.00-15.00 hrs). All testing wasperformed in a normally lighted room. Each test chamber (i.e. activitytest cage) was placed in a soundproofed wooden box with 12 cm thickwalls and front panels and had a dimmed lighting.

Behavioural measurements and apparatus: An automated device, consistingof macrolon rodent test cages (40×25×15 cm), each placed within twoseries of infra-red beams (at two different heights, one low and onehigh, 2 and 8 cm, respectively, above the surface of the sawdust, 1 cmdeep), was used to measure spontaneous and/or drug-induced motoractivity of 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP) andcontrol mice. The following parameters were measured: LOCOMOTION wasmeasured by the low grid of infrared beams. Counts were registered onlywhen the mouse in the horizontal plane, ambulating around the test-cage.REARING was registered throughout the time when at least one high levelbeam was interrupted, i.e. the number of counts registered wasproportional to the amount of time spent rearing. TOTAL ACTIVITY wasmeasured by a sensor (a pick-up similar to a gramophone needle, mountedon a lever with a counterweight) with which the test cage was constantlyin contact. The sensor registered all types of vibration received fromthe test cage, such as those produced both by locomotion and rearing aswell as shaking, tremors, scratching and grooming.

Behavioral measurements (locomotion, rearing and total activity): Twelvedays after MPTP injections (2×40 mg/kg, s.c., 24 hr interval), the micewere administered orally with the different compounds at 3 mg/kg orvehicle (0.1% Tween-80 in 1% methylcellulose) and immediately thereafterplaced in the activity test chambers and their motor behaviors weremonitored for 60 min. After 60 min, the mice were injected with 5 mg/kgL-Dopa (s.c) and then replaced in the test chamber and activitymeasurements maintained for an additional 300 min.

Table 2 presents the locomotion, rearing and total activity counts ofMPTP-treated and control mice administered either tested substances orvehicle administered with a sub threshold dose of L-Dopa.

TABLE 2 TOTAL TREATMENT LOCOMOTION REARING ACTIVITY Vehicle 100%  100% 100%  MPTP + vehicle 16% 25% 46% MPTP + N-(4- 16% 24% 46% bromo-2-nitrophenyl)-5- methoxytryptamine MPTP + 2-(2- 17.6%   25% 45%aminobenzoyl)-N-2- nitrophenyl- ethylamine MPTP + N- 16% 25% 45%(2-carboxy-4- nitrophenyl)- tryptamine MPTP + N-(3,4- 18% 25% 47%dicyanophenyl)- tryptamine MPTP + N-(3,4- 19% 27% 48% dicyanophenyl)-5-methoxytryptamine MPTP + 2-(2- 17% 29% 46% aminobenzoyl)-N-2-nitro-4-cyano- phenylethylamine MPTP + N-(4- 22% 30% 46% Carboxy-2-nitrophenyl)-5- methoxytryptamine MPTP + N-(4- 41% 74% 70%methylsulfonyl-2- nitropheny)-5- methoxytryptamine MPTP + N-(2- 44% 90%73% nitrophenyl)-5- methoxytryptamine MPTP + N- 52% 98% 73% (2-cyano-4-nitrophenyl)- tryptamine MPTP + 2-(2- 54% 100%  72% aminobenzoyl)-N-2-nitro-4- methylsulfonyl- phenylethylamine MPTP + N-(2- 53% 100%  90%Nitrophenyl)- tryptamine2(2-aminobenzoyl)-N-2-nitro-4-methylsulfonyl-phenylethylamine,N-(4-methylsulfonyl-2-nitrophenyl)-5-methoxytryptamine,N-(2-nitrophenyl)-5-methoxytryptamine, N-(2-nitrophenyl)-tryptamine andN-(2-cyano-4-nitrophenyl)-tryptamine (3 mg/kg) significantly reversedthe motor deficits of MPTP-treated mice when combined with asub-threshold (inactive) dose of L-Dopa.Experiment 3:Electrophysiological Characterisation of NMDA-Activated Currents inFreshly Isolated Hippocampal Neurones of Rat.

Isolation of hippocampal neurons: Wistar rats (12-14 days) weredecapitated without anesthesia and the hippocampus was removed. It wasmanually cut into slices (0.2-0.4 mm), in a solution containing (mM):150 NaCl; 5 KCL; 1.25 NaH₂PO₄; 2 CaCl₂; 2 MgCl₂; 26 NaHCO₃; 20 glucose.Slices were preincubated in this solution for 30 min at roomtemperature. The enzymatic treatment proceeded in the same solution withlower Ca²⁺ concentration (0.5 mm) containing 0.4 mg/ml protease fromaspergillus oryzae. The incubation in the enzyme solution proceeded at32° C. within 10 min. Slices were kept subsequently in enzyme-freesolution containing normal Ca²⁺ concentration and used within 6-8 h forobtaining isolated neurons. Throughout the entire procedure thesolutions were continuously saturated with a 95% O₂ and 5% CO₂ gasmixture to maintain pH of 7.4. For cell dissociation the slice wastransferred into the extracellular solution containing (mM): 150 NaCl; 5KCl; 2 CaCl₂; 10 n-2-hydroxyethylpiperazine-n′-2-ethanesulphonic acid(Hepes); pH adjusted with NaOH to 7.4. Single cells were isolated fromCA and CA3 zones of hippocampal slices by vibrodissociation method. Theyhad a diameter 10-15 μm and preserved a small part of dendritic tree.After isolation they were usually suitable for the recording for 1-2 h.

Salines and chemicals: The contents of the extracellular solution was asfollows (in mM): 130 NaCl, 5KCl, 2CaCl₂, 20n-2-hydroxyethylpiperazine-n′-2-ethansulfonic acid (Hepes); 0.1 μm TTX,10 μm glycine, 300 mm 1-aspartate; pH was adjusted with NaOH to 7.4.

The contents of the intracellular solution were as follows (in mM):110CsF, 20Tris-HCl (pH=7.2). L-aspartate and glycine solutions wereprepared on the day of experiment.

The tested substances were dissolved in DMSO.

Current recording and data analysis: The drug-containing solutions wereapplied by the fast “concentration clamp” method using “jumping table”set-up. The currents were recorded with patch clamp technique in thewhole-cell configuration. Recording of the currents was performed usingEPC-7 L/M patch-clamp amplifier.

NMDA-activated currents: The currents were filtered at 3 kHz (three-poleactive Bessel filter) digitally sampled at the rate 6000 μs per pointfor NMDA activated currents. NMDA-induced transmembrane currents weremeasured in the presence of 10 μM glycine and 300 μM L-aspartate in thecontrol and test solutions. The currents were recorded at holdingpotential −70 mV.

Calculations: The inhibition of current at 1 μM of the substance wasaveraged at least for 4 cells. The effect of substance was measured asthe mean ratio I/Io where I was the current under the action ofsubstance and Io was the current in control conditions.

The action of 1 μM tested substances on NMDA-activated currents areshown in Table 3.

TABLE 3 Tested Substance % Inhibition (1 μM) Peak Current Steady StateCurrent N-(3,4-dicyanophenyl)-  93.4%  66.4% tryptamine N-(2- 90.93%66.83% Nitrophenyl)- tryptamine N-(2-Carboxy-4-  76.7%  70.4%nitrophenyl)- tryptamine N-(4-Carboxy-2- 89.92% 74.14% nitrophenyl)-5-methoxytryptamine N-(2-   78%  77.3% Nitrophenyl)-5- methoxytryptamineN-(4-bromo-2- 104.2% 78.18% nitrophenyl)-5- methoxytryptamineN-(3,4-dicyanophenyl)-5-  84.4%  78.4% methoxytryptamine N-(2-cyano-4- 83.1% 82.44% nitrophenyl)- tryptamine N-(2-nitro-4-  95.8%   83%bromophenyl)- tryptamine N-(2-bromo-4- 91.53% 86.57% nitrophenyl)-5-methoxytryptamine 2-(2-aminobenzoyl)-N- 85.95% 87.24% 2-nitro-4-cyano-phenylethylamine N-(2-cyano-4- 96.21% 88.21% nitrophenyl)-5-methoxytryptamine 2-(2-aminobenzoyl)-N- 90.85% 88.77% 2-nitro-4-bromo-phenylethylamine 2-(2-aminobenzoyl)-N- 86.23% 90.78% 2-bromo-4-nitro-phenylethylamine N-(4-Carboxy-2- 101.6% 92.84% nitrophenyl)- tryptamineN-(2-Carboxy-4-   84%  93.6% nitrophenyl)-5- methoxytryptamineThis experiment revealed that N-(3,4-dicyanophenyl)-tryptamine,N-(2-nitrophenyl)-tryptamine, N-(2-carboxy-4-nitrophenyl)-tryptamine,N-(4-carboxy-2-nitrophenyl)-5-methoxytryptamine,N-(2-nitrophenyl)-5-methoxytryptamine,N-(4-bromo-2-nitrophenyl)-5-methoxytryptamine,N-(3,4-dicyanophenyl)-5-methoxytryptamine,N-(2-cyano-4-nitrophenyl)-tryptamine andN-(2-nitro-4-bromophenyl)-tryptamine have a significant blockingactivity on NMDA-activated currents.

1. A compound selected from (a)N-(4-methylsulfonyl-2-nitrophenyl)-5-methoxytryptamine, (b)N-(2-bromo-4-nitrophenyl)-5-methoxytryptamine, (c)N-(4-bromo-2-nitrophenyl)-5-methoxytryptamine, (d)N-(2-cyano-4-nitrophenyl)-5-methoxytryptamine, (e)N-(4-Carboxy-2-nitrophenyl)-tryptamine, (f)N-(2-Carboxy-4-nitrophenyl)-tryptamine, (g)N-(2-Nitrophenyl)-5-methoxytryptamine, (h)N-(4-Carboxy-2-nitrophenyl)-5-methoxytryptamine, (i)N-(2-Carboxy-4-nitrophenyl)-5-methoxytryptamine, (j)N-(2-cyano-4-nitrophenyl)-tryptamine, (k)N-(2-nitro-4-bromophenyl)-tryptamine, (l)N-(3,4-dicyanophenyl)-tryptamine, and (m)N-(3,4-dicyanophenyl)-5-methoxytryptamine.
 2. A pharmaceuticalformulation containing as an active substance a therapeuticallyeffective amount of a compound of claim 1 in association with one ormore pharmaceutically acceptable diluents, preservatives, solubilizers,emulsifiers, adjuvants, excipients or carriers.
 3. The pharmaceuticalformulation of claim 2, which is characterized by at least one of thefollowing features: (i) it is adapted for oral, rectal, parenteral,transbuccal, intrapulmonary, intranasal, topical or transdermaladministration; (ii) it is in unit dosage form, each unit dosagecomprising an amount of said at least one member which lies within therange of about 0.001-about 100 mg/kg body weight; (iii) it is acontrolled release formulation, wherein said at least one member isreleased at a predetermined controlled rate.
 4. The pharmaceuticalformulation of claim 2, which is suitable for oral administration andeach unit dosage comprises from about 0.5 mg to about 50 mg.
 5. Thepharmaceutical formulation of claim 2, which is suitable for parenteralor transdermal administration and each unit dosage comprises from about0.1 mg to about 50 mg.